The broad, long term objectives of the proposed research are to produce folded and rigidified versions of the HIV envelope glycoprotein gp120 for use as potential vaccine candidates. Earlier attempts to use gp120 as a vaccine failed to generate antibodies capable of neutralizing primary isolates of the virus. Antibody responses in vaccinated individuals are often directed against linear epitopes accessible in denatured gp120 that are not exposed in correctly folded gp120. Recently, the structure of core gp120 in complex with a two domain CD4 fragment and the antigen binding fragment of a neutralizing antibody was determined. This structure will be used as a starting point for the design of folded, rigidified, versions of gp120 that should be stable in the absence of CD4 and neutralizing antibody. The specific aims of the project are as follows: 1. Rational design of stabilized mutants of gp120: Analysis of the X-ray crystallographic coordinates of gp120 has been used to devise three different approaches to rigidify the protein. (i) mutations that fill in several large cavities in the structure. (ii) replacements of some Pro and Gly residues identified to be destabilizing based on stereochemical criteria, and replacements of isolated, buried, charged residues. (iii) introduction of disulfide bonds at selected sites. Recombinant proteins will be expressed and preliminary characterization and CD4 binding studies will be attempted. 2. Formation of a stable, folded gp120:CD4 complex: Based on the known crystal structure of the noncovalent gp120: CD4 complex, mutations will be introduced to facilitate disulfide bond formation between gp120 and CD4. In addition, cavities present at the interface will be filled in through appropriate amino acid substitutions in both gp120 and CD4. The mutant gp120 and CD4 proteins will be expressed and formation of a stable, folded covalent complex between gp120 and CD4 will be attempted. Such a stable complex is an attractive vaccine candidate.